Dense core granules are vesicles that store and release bioactive peptides, and are found throughout most eukaryotes. Over the past two decades, detailed models have been developed to explain protein targeting to secretory granules. The most fundamental aspect of these models is that protein sorting in this pathway does not rely on canonical transmembrane receptors or receptor adaptors. However, recent experiments in mammalian neurons led to the surprising finding that bone-derived neurotropic factor (BDNF), a peptide important for neuronal maintenance, is targeted to secretory granules in a pathway, as yet undescribed, that involves a classical lysosomal sorting receptor, sortilin. Moreover, sortilin-family receptor variants have been identified as risk factors for diabetes, and recent work suggests they play a direct role in formation of insulin-containing granules. Another challenge came from recent studies in Drosophila, and in mammalian neuroendocrine cells, which implicated the AP-3 adaptor complex in secretory granule formation. No current model explains the sortilin or AP-3 data. Secretory granules are a prominent feature in the ciliate Tetrahymena thermophila. By studying gene expression during periods of active granule synthesis, it was recently discovered that the Tetrahymena sortilin homologs are highly upregulated under these conditions, in combination with a set of genes that are proposed to underlie secretory granule formation. Those gene products include putative sortilin ligands as well as AP-3 adaptors and other cytoplasmic machinery that is likely to be involved in sortilin trafficking. Preliminary data validate the value of this gene expression-based approach to uncovering mechanisms involved in granulogenesis, and demonstrate that sorting of major classes of granule proteins in Tetrahymena requires sortilins. The proposal is therefore aimed at understanding how sortilins facilitate protein targeting to granules, and what other cellular machinery is involved. To that end, the aims of this proposal include the analysis of sortilin ligands and of adapter proteins that interact with sortilin cytoplasmic domains.